The neurotrophins are a family of structurally and functionally related proteins, including Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5) and Neurotrophin-6 (NT-6). These proteins promote the survival and differentiation of diverse neuronal populations in both the peripheral and central nervous systems (Hefti, 1986; Hefti and Weiner, 1986; Levi-Montalcini, 1987; Barde, 1989; Leibrock et al., 1989; Maisonpierre et al., 1990; Rosenthal et al., 1990; Hohn et al., 1990; Gotz et al., 1994; Maness et al., 1994) and are involved in the pathogenesis of diverse neurological disorders. Neurotrophins exert certain of their biological effects through specific interactions with a class of transmembrane receptor tyrosine kinases (TrkA, TrkB and TrkC) (Kaplan et al., 1991; Klein et al., 1991, 1992; Soppet et al., 1991; Squinto et al., 1991; Berkemeier et al., 1991; Escandon et al., 1993; Lamballe et al., 1991). Specificity of neurotrophin action results from their selective interactions with Trk. That is, TrkA only binds NGF (Kaplan et al., 1991; Klein et al., 1991); TrkB binds BDNF, NT-3 and NT-4/5 (Soppet et al., 1991; Squinto et al., 1991; Berkemeier et al., 1991; Escandon et al., 1993; Lamballe et al., 1991; Klein et al., 1992; Vale and Shooter, 1985; Barbacid, 1993); and TrkC exclusively binds NT-3 (Lamballe et al., 1991; Vale and Shooter, 1985). This is particularly evident when the Trk receptors are coexpressed with the common neurotrophin receptor p75.sup.NTR (For review see Meakin and Shooter, 1992; Barbacid, 1993; Chao, 1994; Bradshaw et al., 1994; Ibanez, 1995).
The common neurotrophin receptor p75.sup.NTR is a transmembrane glycoprotein structurally related to the tumor necrosis factor and CD-40 receptors (Meakin and Shooter, 1992; Ryden and Ibanez, 1996). As all neurotrophins bind to p75.sup.NTR with similar affinity (Rodriguez-Tebar et al., 1990; Hallbook et al., 1991; Rodriguez-Tebar et al., 1992; Ibanez, 1995), neurotrophin specificity is conventionally thought to be caused by the binding selectivity for Trk receptors which are differentially expressed in different neuronal populations (Ibanez, 1995). However, accumulated experimental data on neurotrophin activity reveal important functional aspects of p75.sup.NTR (Heldin et al., 1989; Jing et al., 1992; Herrmann, 1993; Barker and Shooter, 1994; Dobrowsky et al., 1994, Matsumoto et al., 1995; Marchetti et al., 1996; Washiyama et al., 1996). The common neurotrophin receptor enhances functions and increases binding specificity of Trk receptors (Barker and Shooter, 1994; Mahadeo et al., 1994; Chao and Hempstead, 1995; Ryden and Ibanez, 1996). In addition, p75.sup.NTR possesses unique, Trk-independent signalling properties which involve ceramide production through activation of the sphingomyelin cycle (Dobrowsky et al., 1994), apoptosis (cell death) (Van der Zee et al., 1996; Cassacia-Bonnefil et al., 1996; Frade et al., 1996), and activation of the transcription factor NFKB (Carter et al., 1996). Recently, p75.sup.NTR has been demonstrated to participate in human melanoma progression (Herrmann et al., 1993; Marchetti et al., 1996). Furthermore, NGF and NT-3 increase the production of heparin by 70W melanoma cells, which is associated with their metastatic potential (Marchetti et al., 1996). Although this effect has been shown to be mediated by the common neurotrophin receptor, neither BDNF nor NT-4/5 appeared to be active. Explicit modelling of the various neurotrophin receptors will be critical to the rational design of neurotrophin-based drugs.
NGF displays high and low affinity binding sites in sensory and sympathetic neurons and in pheochromocytoma PC12 cells (Sutter et al., 1979; Landreth and Shooter, 1980; Schechter and Bothwell, 1981). The coexpression of the common neurotrophin p75.sup.NTR receptor with TrkA is required to form the high affinity binding site (Hempstead et al., 1991; Barker and Shooter, 1994; Mahadeo et al., 1994; Chao and Hempstead, 1995). Several models of the TrkA-p75.sup.NTR interaction have been proposed to explain high affinity NGF binding (Bothwell, 1991; Chao, 1992b; Chao and Hempstead, 1995; Wolf et al., 1995; Ross et al., 1996; Ross et al., 1997). These models differ with respect to direct (conformational model) or indirect (ligand-presentation model) interaction of p75.sup.NTR with TrkA. Direct TrkA-p75.sup.NTR interaction is consistent with much of the existing experimental data.
NGF, a 118 amino acid protein, is an extremely important neurotrophin, being implicated in the pathogenesis of Alzheimers disease, epilepsy and pain (Ben and Represa, 1990; McKee et al., 1991; Leven and Mendel, 1993; Woolf and Doubell, 1994; Rashid et al., 1995; McMahon et al., 1995). The binding of NGF to its receptors is determined by distinct sequences within its primary amino acid structure. While several regions of NGF participate in the NGF/TrkA interaction, mutation studies suggest that relatively few key residues, namely those located in the NGF amino and carboxyl termini, are required for high affinity binding.
The hairpin loop at residues 29-35 is responsible for recognition by p75.sup.NTR (Ibanez et al., 1992; Radziejewski et al., 1992), while the amino and carboxyl termini are important binding determinants for recognition by the TrkA receptor (Shih et al., 1994; Moore and Shooter, 1975; Suter et al., 1992; Burton et al., 1992, 1995; Kahle et al., 1992; Luo and Neet, 1992; Drinkwater et al., 1993; Treanor et al., 1995; Taylor et al., 1991). Truncation of either the amino or carboxyl terminus of NGF produces less active NGF analogues; similarly most deletion or point mutations of the amino terminus also lead to NGF analogues with diminished activity (Shih et al., 1994; Burton et al., 1992, 1995; Kahle et al., 1992; Drinkwater et al., 1993; Treanor et al., 1995; Taylor et al., 1991). On the other hand, the NGF.DELTA.2-8 (NGF with residues 2-8 removed) and NGF.DELTA.3-9 deletion mutants are almost as active as wild type NGF (Drinkwater et al., 1993). These NGF structure-activity relationships in combination with the considerable species variability (mouse, human, guinea pig and snake) of the amino acid sequence of the NGF termini (McDonald et al., 1991) are of potential value in understanding the NGF/TrkA interaction.
NGF exerts its biological activity as a non-covalent dimer (Treanor et al., 1995; Burton et al., 1995; McDonald et al., 1991; Ibanez et al., 1993; Bothwell and Shooter, 1977). Two 118 residue NGF monomers are dimerized by hydrophobic and van der Waals interactions between their three anti-parallel pairs of .beta.-strands; consequently, the amino terminus of one NGF monomer and the carboxyl terminus of the other are spatially juxtaposed (McDonald et al., 1991). Furthermore, although a dimer has 2 pairs of termini, only one pair of termini is required for TrkA receptor recognition (Treanor et al., 1995; Burton et al., 1995). Accordingly, solving the conformation of the complex formed by the amino terminus of one monomer and the carboxyl terminus of the other in dimeric NGF is of fundamental relevance to understanding the interaction of NGF with TrkA.
The X-ray crystallographic 3-dimensional structure of a dimeric mouse NGF (mNGF) has been reported recently (McDonald et al., 1991). However, within this structure, the amino terminus (residues 1-11) and the carboxyl terminus (residues 112-118) remain unresolved for both pairs of termini. High flexibility of the NGF termini makes it difficult to experimentally determine their bioactive conformations, particularly since transition metal ions commonly used in X-ray crystallography (McDonald et al., 1991) have high affinity for His residues (Gregory et al., 1993) which are present in the NGF amino terminus (Bradshaw et al., 1994). Indeed, conformational alterations in the receptor binding domains of NGF caused by Zn.sup.2+ cations leading to its inactivation have been described recently (Ross et al., 1997). Since the amino and carboxyl termini are crucial for NGF bioactivity as mediated via TrkA and because of the significance of NGF in multiple neurologic disease processes, the determination of the biologically active conformation of these termini is an important and challenging problem for computational chemistry.
In contrast with NGF, little is known about the BDNF binding determinant for TrkB activation. Although there were several attempts to identify structural elements of BDNF that determine its receptor specificity (Ibanez et al.,1991, 1993,1995; Suter et al., 1992; Ilag et al., 1994; Kullander and Ebendal., 1994; Urfer et al., 1994; Lai et al., 1996), the results of mutational experiments are still controversial. Thus, the reported importance of residues 40-49 (variable loop region 11) for TrkB related activity of BDNF (Ibanez et al., 1991; Ilag et al., 1994) has not been confirmed by recent studies (Lai et al., 1996), in which it has also been shown that the key residues of the TrkB receptor binding domain of BDNF are situated between residues 80 and 109. The latter result is consistent with earlier observations that the BDNF termini are not involved in TrkB binding and that the key residues of the BDNF binding epitope are Arg.sup.81 and Gln.sup.84 (Ibanez et al., 1993). In addition, it has been found that receptor binding domains of BDNF and NT-3 are located in the same regions of the molecules, i.e. in their "waist" parts (Ibanez et al., 1993; Urfer et al., 1994). As far as the NT-3 receptor binding domain is concerned, detailed mutational studies carried out by Urfer et al. (1994) revealed that it comprises a surface that includes major parts of the central .beta.-strand stem, with the most important residues being Arg.sup.103, Thr.sup.22, Glu.sup.54, Arg.sup.56, Lys.sup.80 and Gln.sup.83. These residues correspond to Arg.sup.104, Thr.sup.21, Glu.sup.55, Lys.sup.57, Arg.sup.81 and Gln.sup.84 of BDNF, respectively (McDonald et al., 1991).
The location of the neurotrophin binding sites within the Trk receptors is the subject of debate (MacDonald and Meakin, 1996). Published data indicate the existence of two putative neurotrophin binding sites of Trk proteins: the "second immunoglobulin-like domain" (residues Trp.sup.299t -Asn.sup.365t, denoted IgC2) and the "second leucine-rich motif" (LRM-2A and LRM-2B, residues Thr.sup.97 -Leu.sup.120 of TrkA and TrkB, respectively) (Schneider and Schweiger, 1991; Kullander and Ebendal, 1994; Perez et al., 1995; Urfer et al., 1995; Windisch et al., 1995a, 1995b, 1995c; MacDonald and Meakin, 1996; Ryden and Ibanez, 1996).
p75.sup.NTR belongs to a family of cell surface proteins that share a common pattern of four repeated cysteine-rich domains (CRDs) in the extracellular portion (Yan and Chao, 1991; Baldwin and Shooter, 1994). X-ray crystallographic studies on the extracellular portion of the related protein p55.sup.TNFR revealed that CRDs fold independently of each other, and three conserved disulfide bonds maintain a specific geometry of each CRD which consists of two loops (Banner et al., 1993) (loops A and B, FIG. 1a). Very little is known about the p75.sup.NTR receptor functional epitope and, particularly, about residues directly participating in molecular recognition processes. All four CRD repeats of p75.sup.NTR are found to be required for binding, with the second CRD being most important (Baldwin and Shooter, 1995). Residue Ser.sup.50 of p75.sup.NTR seems to be essential for NGF binding (Baldwin and Shooter, 1995).
The results of site-directed mutagenesis suggest that the p75.sup.NTR receptor binding domains within neurotrophins are juxtaposed positively charged residues located in two adjacent hairpin loops which represent variable region I (residues 23-35) and V (residues 93-98) (Ibanez et al., 1992; Ryden et al., 1995; Ibanez, 1994; Ibanez, 1995; Ryden and Ibanez, 1996) (FIG. 1c). In NGF, residues Lys.sup.32, Lys.sup.34, and Lys.sup.95 have been found to be involved in p75.sup.NTR receptor binding, with Lys.sup.32 making the strongest contact, followed by Lys.sup.34 and Lys.sup.95. In addition, some role of Asp.sup.30, Glu.sup.35, Arg.sup.103, Arg.sup.100, Lys.sup.88, and Ile.sup.31 in p75.sup.NTR binding and biological activity has been demonstrated (Ibanez et a., 1992; Ibanez, 1994). Two residues of variable region I, namely Arg.sup.31 and His.sup.33, have been demonstrated to be essential for binding of NT-3 to p75.sup.NTR, whereas similarly located Arg.sup.34 and Arg.sup.36 mediate binding of NT-4/5 (Ryden et al., 1995). In contrast to NGF, a positively charged residue in variable region V is not critical for binding of NT-3 or NT-4/5 to p55.sup.NTR (Ryden et al., 1995). In BDNF, however, only residues of variable region V, Lys.sup.95, Lys.sup.96 and Arg.sup.97, bind to p75.sup.NTR and compensate for the lack of positively charged residues in variable loop region I (Ryden et al., 1995).
Therefore, it is an object of the invention to establish explicit atom-level models of the Trk and p75.sup.NTR recognition sites for neurotrophins.
It is a further object of the invention to provide atom-level models of the mechanism of interaction of neurotrophins and their receptors.